1. Field of the Invention
Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the .sup.32 p labelling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
One method for detecting specific nucleic acid sequences generally involves immobilization of a target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labelled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
When very low concentrations must be detected, such a method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable.
Recently, a method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
Another method has also recently been described for amplifying nucleic acid sequences. This method is referred to as single primer amplification. The method provides for the amplification of a target sequence that possesses a stem-loop or inverted repeat structure where the target sequence is flanked by relatively short complementary sequences. Various methods for creating such a target sequence in relation to the presence of a polynucleotide analyte to be detected have also been described.
The above methods are extremely powerful techniques for high sensitivity detection of target DNA molecules present in very small amounts. The correlation between the number of original target DNA molecules and the number of specifically amplified products is influenced by a number of variables. Minor variations in buffer or temperature conditions can greatly influence reaction-to-reaction amplification efficiencies. Further, clinical samples of DNA targets can contain inhibitory factors that can suppress enzymatic amplification.
When amplifying a target sequence of a nucleic acid for use in clinical diagnostics, there is a need to assure that each amplification reaction is capable of yielding an amplified product. In particular commercial diagnostic products require validation measures to avoid misdiagnosis due to improper assay methods or contaminated or inactive reagents. Of importance is the development of an internal positive control for demonstrating that the reagents and the detection methodology are working properly. Without such a control the failure of an assay to show the presence of a target nucleic acid sequence may be due to the absence of the target or may be caused by a failure of one or more reagents or of an instrument used in conducting an assay.
Various approaches have been developed for qualification or quantitation of amplification reactions and these approaches can be divided into two main categories, namely, homologous controls and heterologous controls. Such controls have been applied to amplification of mRNA and adapted for DNA analytes. Heterologous controls have a control polynucleotide that does not contain target sequences. One such approach is known as the "endogenous standard" assay, which utilizes as a standard an endogenous polynucleotide that is expressed at a relatively constant level in all samples to be tested. The level of the test sequence is then compared to the standard. Heterologous controls are commonly amplified regions of human DNA such as HLA-DQ and beta-globin genes or mRNA. Heterologous controls assure the adequacy of all the non-target specific reagents and the procedure but are insensitive to any problem involving a target-specific reagent.
Homologous controls utilize a control polynucleotide that contains some of the same sequences as the intended target, but is distinguishable from the target by a difference in size or by the presence or absence of a unique sequence such as a restriction site. Homologous controls contain exogenous nucleic acid fragments, i.e., they are not naturally present in a sample, and they are constructed so that they can be amplified with the same primers used to amplify the target. In this approach a synthetic standard is designed to have only slight variations in sequence but readily distinguishable from a target sequence. The sample to be assayed and the synthetic standard are amplified in the same reaction vessel and any variable that may affect amplification should affect both the target and the control equally.
Generally, in the above methods there is a competition between amplification of the control and the target if present, such as competing for binding to primers and for the other reagents such as nucleoside triphosphates and polymerase. The competition results usually because of the availability of only a limited amount of the polymerase. As a result the presence of a high concentration of one of these species can block amplification of the other and thus potentially interfere with detection of either the control or the target. Thus, for example, in order to achieve co-amplification of two DNA species of similar size in PCR, it is usually necessary to begin the amplification with nearly equal concentrations of the two DNA target sequences.
2. Description of the Related Art
U.S. Pat. No. 5,219,727 (Wang, et al.) discusses a method for determining the amount of a target nucleic acid segment in a sample by polymerase chain reaction. The method involves the simultaneous amplification of the target nucleic acid segment and an internal standard nucleic acid segment. The amount of amplified DNA from each segment is determined and compared to standard curves to determine the amount of the target nucleic acid segment present in the sample prior to amplification. The method has particular applicability for determining the quantity of a specific mRNA species in a biological sample. This development is also discussed by Wang, et al., in Proc. Nat. Acad. Sci. USA (1989) 86:9717-9721.
Quantitative PCR methods are disclosed by Eeles, et al., in "Polymerase Chain Reaction (PCR): The Technique and Its Applications" (1993) Chapter 6, pages 55-61, R.G. Landes Company.
The elimination of false negatives in nucleic acid amplification is discussed in European Patent Application No. WO 94/04706 (Kievits, et al.). Prior to amplification an internal control is added to the sample. The control has a nucleic acid distinguishable from the analyte nucleic acid that can be amplified with the same amplification reagents as the analyte nucleic acid, preferably a nucleic acid sequence corresponding to the analyte nucleic acid that has been mutated to discriminate it from the analyte nucleic acid.
Celi, et al., describe a rapid and versatile method to synthesize internal standards for competitive PCR in Nucleic Acids Research (1993) 21(4):1047.
Gilliland, et al., discuss the analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction in Proc. Natl. Acad. Sci. USA (1990) 87:2725-2729.
PCR mimics:competitive DNA fragments for use as internal standards in quantitative PCR are disclosed by Siebert, et al., in Biotechniques (1993) 14(2):244-249.
Piatak, et al., describe quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species in Biotechnicues (1993) 14(1):70-80.
Quantitative PCR and RT-PCR in virology is disclosed by Clementi, et al., in PCR methods and Applications (1993) 2:191-196.
Competitive polymerase chain reaction using an internal standard: application to the quantitation of viral DNA is discussed by Telenti, et al., Journal of Virological Methods (1992) 39:259-268.
A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by polymerase chain reaction is described by Saiki, et al., (1986) Science, 230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al., Science (1988) 239:487.
U.S. patent applications Ser. Nos. 07/299,282 filed Jan. 19, 1989, now U.S. Pat. No. 5,508,178, describe nucleic acid amplification using a single polynucleotide primer (ASPP). U.S. patent application Ser. No. 07/555,968 filed Jul. 19, 1990, now U.S. Pat. No. 5,439,998 (Aug. 8, 1990), describes a method for producing a molecule containing an intramolecular base-pair structure. A method for introducing defined sequences at the 3'-end of a polynucleotide is described in U.S. patent application Ser. No. 08/140,369, filed Oct. 20, 1993. The disclosures of these five applications are incorporated herein by reference including the references listed in the sections entitled "Description of the Related Art."
Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers is described in U.S. Pat. No. 5,043,272. A single stranded self-hybridizing nucleic acid probe capable of repeatedly hybridizing to itself or other nucleic acids to form an amplified entity is described in U.S. patent application Ser. No. 07/888,058, filed Jul. 22, 1986.